Introduction of a stabilizing 10 residue {beta}-hairpin in Bacillus subtilis neutral protease |
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Authors: | Eijsink, Vincent G.H. Vriend, Gerrit van den Burg, Bertus van der Zee, J.Rob Veltman, Oene R. Stulp, Ben K. Venema, Gerard |
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Affiliation: | Department of Molecular Genetics, Center for Biological Sciences, University of Groningen Kerklaan 30, 9751 NN Haren, The Netherlands 1EMBL, BIOcomputing Program Meyerhofstrasse 1, D-6900 Heidelberg, Germany |
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Abstract: | A 10 residue ß-hairpin, which is characteristic ofthermostable Bacillus neutral proteases, was engineered intothe thermolabile neutral protease of Bacillus subtilis. Therecipient enzyme remained fully active after introduction ofthe loop. However, the mutant protein exhibited autocatalyticnicking and a 0.4°C decrease in thermostability. Two additionalpoint mutations designed to improve the interactions betweenthe enzyme surface and the introduced ß-hairpin resultedin reduced nicking and increased thermostability. After theintroduction of both additional mutations in the loopcontainingmutant, nicking was largely prevented and an increase in thermostabilityof 1.1°C was achieved. |
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Keywords: | Bacillus/ ß -hairpin/ neutral protease/ thermostabilit |
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